1. Field of the Invention
The present invention relates to a process to remove cholesterol from dairy products. More specifically the present invention relates to a process of contacting a dairy product, such as milk, cream or butteroil, with saponin at an elevated temperature, followed by treatment with at least one diatomaceous earth or other adsorbent, and filtering to remove the insoluble cholesterol-saponin obtained.
2. Description of the Related Art
Cholesterol present in a variety of foods including meat and dairy products has long been implicated as a source of the high levels of cholesterol in humans. Milkfat, for example, usually contains about 310 milligrams total cholesterol per 100 grams (Christie, 1983). Further essentially all of the cholesterol in milk fat is present as free cholesterol with only traces of cholesterol present as an ester.
Therefore, it would be desirable to have an inexpensive large-scale process to remove cholesterol from dairy products ultimately used as foodstuffs.
Methods are known to remove cholesterol from dairy products. These include, for example, steam stripping, supercritical fluid extraction (SFE) using carbon dioxide, specific enzymatic cholesterol reductase or, adsorption using cyclodextrins. However, these other methods are lengthy, equipment intensive and/or expensive.
The following references are of general and specific interest:
AOAC Official Methods of Analysis, 14th Ed. ".beta.-Sitosterol in butteroil." p. 522. Assoc. Off. Anal. Chem , Arlington, Va. PA0 W. W. Christie, (1983). "The composition and structure of milk lipids. p.5 in Developments in Dairy Chemistry, Vol. II. Applied Science. New York, N.Y. PA0 A. M. Glaurert, et al, (1962). "Action of saponin on biological cell membranes." Nature Vol. 194:953-955. PA0 M. KATES, et al (1972). "Total and free cholesterol." in Techniques of Lipidology, P. 360. American Elsevier, New York, N.Y. PA0 I. Katz, et al. (1967). "Rapid method for isolation of unesterified sterols and its application to detection of milkfat adulteration with vegetable oils." J. Dairy Sci., Vol. 50:1764-1768. PA0 K. R. Price, et al. (1987). "The chemistry and biological significance of saponins in foods and feeding stuffs." CRC Critical Reviews in Food Science and nutrition. Vol. 26:27-135. PA0 D. P. Schwartz, et al. (1967). "Rapid quantitative procedure for removing cholesterol from butterfat." J. Lipid Res. Vol. 8:54-59. PA0 P. Seeman, (1974). "Ultrastructure of membrane lesion in immune lysis, osmotic lysis and drug-induced lysis." Federal Proceedings. Vol. 33:2116-2120. PA0 S. Takagi, et al. (1982). "Digitonin-cholesterol complex formation: effects of varying the length of the side chain. Chem. Phar. Bull. 30:3485-3491. PA0 D. P. Schwartz, et al., U.S. Pat. No. 3,450,541. PA0 J. Potha, U.S. Pat. No. 4,546,097. PA0 T. E. Furia (ed) (1980) CRC Handbook of Food Additives VII, CRC Press, Boca Ratan, Fla. PA0 W. J. Hurst, et al. (1985), "HPLC Determination of the Cholesterol Content of Egg Noodles as an Indicator of Egg Solids," Journal of Agricultural Food Chemistry, Vol. 33, pp. 820-822. PA0 M. R. Malinow, et al., U.S. Pat. No. 4,242,502 issued Dec. 30, 1980, Class 536/5. PA0 J. Courregelongue et al., U.S. Pat. No. 4,880,573, issued Nov. 14, 1989, Class 260/420. PA0 S. Arichi et al., U.S. Pat. No. 4,524,067, issued Jun. 18 1985, Class 514/33. PA0 F. Uenobe et al., U.S. Pat. No. 4,489,067 issued Dec. 10, 1984, Class 424/195. PA0 European Patent application 318,326 issued May 1989.
All of the references, patents, standards, etc. cited in this application are incorporated by reference in their entirety.
Saponins are glycosides, occurring primarily but not exclusively, in plants that in general share a number of properties, including, binding of 3-.beta.-OH sterols, marked foaming in water, surfactant properties and hemolysis of red blood cells when aqueous solutions are injected into the bloodstream. Saponins occur widely in plants used for food and feed and have a wide variety of structures which are reflected in their varying chemical and biological properties. In general, the aglycone or non-sugar portion of the molecule is a hydrophobic steroidal or triterpenoidal derivative whereas the polar carbohydrate moiety is comprised of different oligosaccharides of varying chain length, some being only mono- or di-saccharides. The sugars associated with the aglycone may include rhamnose, glucose, galactose, xylose, arabinose, glucuronic acid or mixtures thereof. Coupling of the oligosaccharide to the aglycone can involve hemiacetal linkages between the reducing end of a sugar residue or an ester linkage between the carboxyl group of the glucuronate both linked with a hydroxyl group of the aglycone.
The nature of the cholesterol-glycoside complex is apparently not known with certainty. Based on electron micrographs, Seeman (1974) and Glauret et al. (1962) proposed a micellar type arrangement where three saponin molecules associate via the carbohydrate residues projecting inward. The aglycone on the periphery complexes with cholesterol on an equimolar stoichiometry. More recently Takagi et al (1982) have proposed a structural model for the saponin-cholesterol complex based on studies of the interaction between digitonin and a series of cholesterol analogues. The data of Takagi et al. (1982) are not in agreement or compatible with the structure described by Seeman (1974) and Glauret et al. (1962). The data of Takagi et al. (1982) suggest that the digitonin-cholesterol complex is a clathrate in which digitonin molecules associate to form a hydrophobic pocket in which the cholesterol is a guest. Takagi et al. (1982) proved that the stoichiometry of the cholesterol:digitonin complex is 1:1
Many, but not all, saponins are fungistatic. Because they can hemolyze red blood cells, saponins are generally toxic when injected intravenously. However, the oral toxicity of many saponins is quite low because they are not absorbed from the gut. There is some evidence that saponins included in feeds at relatively high levels leads to reduced growth rates in poultry and monogastric mammals. Most common saponins of foods and feedstuffs seem to be free of significant oral toxicity. For example, soybean saponins fed at high concentrations to chicks, mice and rats had no adverse effects. Rats fed alfalfa saponins at a level of 1% in the diet for up to 6 months showed no ill effects, although a potentially beneficial reduction in serum cholesterol and triglycerides was observed. In monkeys (primates also known as Macaca fascicularis), no adverse effects were observed after the consumption of an undefined mixture of alfalfa top saponins for up to 18 months.
The triterpenoid saponins from a soap bark (also known as Ouillaja saponaria) are widely used in some countries as food additive (foaming and emulsifying agent) and have been subjected to thorough toxicological tests. No significant toxic effects were observed in short-term feeding studies in rats, nor in mice fed the material at levels as high as 1.5% over a prolonged period. Saponin-containing extracts of a yucca plant (Yucca mohavensis) were found to be less hemolytic than those of soybeans. No adverse effects of feeding commercial Yucca mohavensis saponin (0.05%) for 12 weeks were noted in rats in respect to growth, food utilization, blood counts, blood glucose and nonprotein N or in gross or histological findings post-mortem.
Retardation of growth by dietary saponins was first observed in chicks fed alfalfa. The effect could be overcome by the addition of 1% cholesterol in the diet; presumably by forming a complex with the saponins in the gut. Growth retardation by dietary saponins has been observed in other avian species and in other monogastric animals (particularly swine) and in laboratory mammals. Isolated saponins from a trefoil (also known as Medicago lupulina) and from other genera, such as Quillaja and Gypsophila species have also been shown to cause growth retardation in rodents. Primates appear to be more resistant to the detrimental biological effects of dietary saponins, and some researchers believe that primates may have adapted to saponins in the diet. It would appear that saponins in foods and feeds have a low order of oral toxicity.
A number of researchers have observed that different saponins exert a hypocholesterolemic effect when included in the diet of various animals at non-toxic levels. (See Price et al. (1987)). The effect appears to be more than binding of cholesterol in the gut to prevent its absorption. Evidence has been presented that saponins also increase the fecal excretion of bile acids. Since bile acids are derived from the body's cholesterol this decreases the body burden of cholesterol, thereby lowering serum cholesterol. The hypocholesterolemic effect of saponins has prompted some biomedical researchers to propose using foods containing them in serum cholesterol-lowering diets.
Saponins occur in a variety of common foods and feeds as well as herbs and tonics (Price et al., 1987). In Table 1 are listed some foods, herbs, flavorings, health foods, tonics and feeds containing substantial concentrations of saponins.
TABLE 1 ______________________________________ Saponins (% where given) in selected foods, herbs, flavorings, health foods, tonics and feeds (See Price, et al., 1987 above). PLANT PRODUCT Saponins, % ______________________________________ Foods Soybeans - full fat 0.22-5.6 dry wt. Protein isolates 0.3-2.5 dry wt. 0.76 wet wt. Defatted soy flour 0.67 wet wt. Fermented soy products 0.25-0.84 dry wt. Tofu 0.30-2.1 dry wt. Butter beans 0.10 dry wt. Kidney beans 0.2-1.6 dry wt. Navy beans 0.4-2.1 dry wt. Canned baked beans 0.45 dry wt. Green pea 0.18-4.2 dry wt. Peanuts 0.001-1.6 dry wt. Asparagus 1.5 dry wt. Garlic 0.3 dry wt. Alfalfa sprouts 8.0-8.7 dry wt Oats 01-0.13 dry wt. Sesame seeds 0.30 dry wt. Tomato seeds 1.0 dry wt. Onions Lentils Green pepper Tea Pumpkins Melons/Watermelons Yams Cucumber Blackberry Herbs, Flavorings, Health Foods, Tonics Aloe Lemongrass Sage Fenugreek Licorice Nutmeg Quillaja Saponaria Yucca Gypsophila Ginseng Feeds Alfalfa 0.17-1.71 Alfalfa meal 1.26 Horse chestnut 3-6 Lupins 1.1-1.7 Black medic trefoil 3.5 ______________________________________
It is apparent from Table 1 that saponins occur in a wide variety of plants, many of them consumed by humans. Also, common animal feeds contain substantial quantities of saponins. Quillaja saponaria (soapbark) which contains 10% saponins (Price et al., 1987) and Yucca mohavenensis (rich in saponins) are evidently approved in the U.S.A. for use as food additives with no apparent limitations (Furia, 1980). Crude extracts of these sources of saponins are commercially available and relatively low in price.
It is reported that an analytical procedure using benzene is described for the removal and determination of cholesterol in milk-fat using digitonin, which is a known regulated toxic cardiac stimulant for mammals, e.g. human beings. (See D. P. Schwartz et al. (1967)). The use of benzene solvent has its difficulties, and high cost and cannot be applied to foodstuffs.
It would therefore be very valuable to use the less expensive crude extracts of plant saponins (food grade) for the large scale removal of cholesterol from dairy products which can be used in food-stuffs. The present invention provides such a procedure.